INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Primary alveolar proteinosis (AP) is a chronic disease of unknown pathogenesis characterized by the diffuse accumulation of excess surfactant in the air spaces. Patients are usually younger than 45 yr of age with an appreciable proportion adolescents and infants (1, 2). Although whole-lung lavage has become standard therapy, the clinical course varies markedly (2).

Although surfactant lipid composition appears altered in patients with AP (1), it is unclear whether these changes are primary or secondary, possibly associated with increased desquamation of epithelial cells. Early studies by Ramirez-Rivera and Harlan (3) and McClenahan and Mussenden (4) suggest that surfactant synthesis and secretion in patients with AP is normal and that accumulation arises through an impairment in surfactant removal.

In the air spaces, surfactant protein-A (SP-A) has a highly organized quaternary structure comprising distinct binding domains for phospholipids, calcium, carbohydrates, glycolipids, and lipopolysaccharides (5). It is evident from its myriad of different isoforms that SP-A must be regarded as a family of related proteins, which, in humans, are derived from more than one gene product (6). Although the roles of these isoforms are unresolved, alveolar SP-A is implicated in surfactant homeostasis (5) and lung host defense (7). Therefore, aberrations in either SP-A structure or abundance could contribute not only to the primary pathogenesis of AP but also to the prevalence of secondary infections (2).

Although structural differences in SP-A have been implicated in AP (8, 9), there has not been a detailed study of the isoforms. Moreover, whereas SP-A is often asserted to be elevated in AP, no one has examined the ratio of surfactant proteins to lipids; this may indicate possible defects in surfactant homeostasis. In this report we present a detailed analysis of surfactant composition in 13 patients with AP. In seven patients lavage fluid from both right and left lung (RL, LL) was available. In one patient, presented as a case study, lavage fluid was collected on 11 consecutive occasions, and composition was examined in each sequential lavage aliquot from the early onset of the condition through to its apparent resolution, ~ 400 d later.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Lavage

Bronchoalveolar lavage of a right middle lobe segmental bronchus was performed on 16 normal subjects using four 20-ml volumes of 0.15 M saline (10). This study was approved by the Ethics Review Committee for Clinical Investigation of the Flinders Medical Centre (Permit no. 83/93).

In 13 patients with AP (six female: 28 yr [16-70 yr]; seven male: 43 yr [17-51 yr]; median [range]) idiopathic AP was diagnosed, both clinically and on the basis of either transbronchial or open lung biopsy. Patients were anesthetized with thiopental sodium and vecuronium bromide (or propofol) and intubated with a left double-lumen endotracheal tube. Anesthesia was maintained with isofluorane (or propofol infusion), fentanyl, and vecuronium bromide, and patients' volume control mechanically ventilated with an FI_O2 of 1. Lavage was performed by gravitational infusion (30 cm H₂O) and drainage of saline (37° C) in sequential aliquots of 1.0 to 1.5 L; the lung was percussed during the procedure. A cumulative total of instilled and drained fluid was maintained to avoid excessive fluid retention. The procedure was terminated when the lavage effluent was essentially clear (usually 1.5 to 2 h and requiring between 20 to 30 L saline). Both lungs were then ventilated and the airways suctioned. Compliance of both lungs was assessed at 10-min intervals, and the lung was deemed recovered when the postlavage matched its prelavage compliance (approximately 20 to 30 min). The other lung was lavaged, usually within a week. Although both lungs were lavaged, in some patients fluid was available only from one side. Lavage aliquots were stored at 70° C for blind, randomized batch analysis.

Surfactant Analysis

After first freeing the SP-A and -B from associated surfactant components, the proteins were measured with ELISA inhibition assays using polyclonal antibodies raised against AP-derived SP-A (Po-A) and mature SP-B, respectively (10).

Lipids were extracted from the lyophilized lavage fluid, phospholipids and disaturated phospholipids (DSP) were separated, and inorganic phosphorus content was determined (10). Samples were corrected back to phospholipid content (× 25) as per usual (10). Phospholipid classes were separated by high performance liquid chromatography (HPLC) (µPorasil Silica; Waters Millipore, Bedford, MA) as described by Pison and associates (11) and quantified using a mass detector (Model 750/14; Applied Chromatography System, Cheshire, UK) coupled to a Delta Chromatography Data System program (Digital Solutions, Margate, Australia), Sphingosine (Sigma Chemicals, St. Louis, MO) was included as an internal standard. Neutral lipids were separated by HPLC using a Waters ¹⁸C Novapak column (Waters Millipore) and free cholesterol (CHOL) was quantified by its absorption at 210 nm (12).

Electrophoresis, Protein Blotting and Immunochemical Staining

Lavage fluid aliquots were lyophilized, delipidated, and desalted (10). Isoelectric focusing (IEF) of samples containing ~ 20 µg of proteins was performed for 35,000 Vh using an Immobiline DryStrip Kit (pH, 4.0 to 7.0) according to the Pharmacia LKB Biotechnology instructions (Uppsala, Sweden). The samples were separated in the second dimension and stained with silver or transferred onto nitrocellulose (10). Molecular weight and carbamylyte IEF standards were obtained from Pharmacia LKB Biotechnology. The blots were developed using Po-A (8 µg/ml) as the primary antibody, alkaline phosphatase-conjugated sheep antirabbit polyclonal IgG (1 µg/ml) (Silenus Laboratories, Melbourne, Australia) as the secondary antibody, and a Protoblot Immunoscreening System (Promega, Madison, WI) (10). We did not study SP-B electrophoretically because of the inherent difficulties in separating the hydrophobic protein by IEF.

Results are expressed as the mean ± SEM. The Mann-Whitney U Test or Wilcoxon's Matched Pairs Sign Rank Test was used for all comparisons. The association between measured variables was tested using Spearman's Rank Order Correlation Test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Surfactant Composition

There was no difference between RL and LL in the relative amounts of any of the surfactant components when compared either as a combined group, the seven patients with matching RL and LL specimens, or the patient from whom lavage fluid was collected on 11 consecutive occasions (six RL and five LL) (Table 1).

When the data were combined, the percentage of phosphatidylcholine (PC) did not differ from that in normal lavage fluid (Table 1). In contrast, the percentages of phosphatidylglycerol and phosphatidylserine, and the phosphatidylglycerol/ phosphatidylinositol ratio (PG/PI) were all decreased (p < 0.01, 0.05, and 0.01, respectively), whereas the percentages of sphingomyelin (SPH) and lysophosphatidylcholine (LPC) were increased (both p < 0.01). The percentage of PI and phosphatidylethanolamine were reduced (both p < 0.01) if data from the 11 consecutive samples from the case study patient were excluded. However, the relative phospholipid composition varied markedly between patients, as reflected in the large SEM.

There was no difference in the percentage of DSP as a proportion of the total phospholipids (PL) (%DSP/PL) in lavage fluid from RL and LL, or in the CHOL/DSP, SP-A/DSP and SP-B/DSP ratios (Table 2). Again, this was true regardless of whether the data were compared either as a combined group, the seven patients for whom matching RL and LL specimens were available, or the patient from whom lavage fluid was collected on 11 consecutive occasions. Although the %DSP/PL did not differ from that in normal lavage, there was considerable variation between patients. The SP-A/DSP and SP-B/ DSP ratios were generally elevated (both p < 0.01); however, again, considerable variation was evident between patients, as reflected in the large SEM. In contrast, whereas the CHOL/ DSP ratio was invariably elevated (p < 0.01), it was remarkably constant between patients.

SP-A Immunochemical Analysis

Normal lavage contained a large number of proteins, the most abundant being albumin and IgG light chains (Figure 1). Under reducing conditions, normal SP-A immunostained as a major series of spots spanning 30 to 36 kD and a pI range of 4.6 to 5.9. In addition, two minor series of spots corresponding to unglycosylated SP-A (28 to 30 kD, 5.7 to 6.1 pI) and SP-A dimer (59 to 67 kD, 4.9 to 5.9 pI) were detected. In AP lavage, the relative abundance of the SP-A isoforms differed from that in normal lavage, and varied markedly from patient to patient, extreme examples of which are illustrated in Figure 1.

View larger version (95K): [in this window] [in a new window]   Figure 1.   Two-dimensional electrophoretic silver (Frame 1) and immunochemical (Frames 2 to 6) staining patterns of pooled normal lavage (Frames 1 and 2) and whole lavages from patients with AP (Frames 3 to 6). Isoelectric focusing (IEF) of the samples was performed simultaneously. The samples were separated under reducing conditions. Mr (Std) and IEF (carbamylated creatine phosphokinase: 34 spots; pI, 4.9 to 7.1) standards are labeled on the silver-stained gel (Frame 1), as is albumin (Alb) and the light chain of immunoglobulin (IgG). Only a selected window of immunochemical staining (Mr ~ 25 to ~ 100 kD and pI ~ 4.2 to ~ 6.4), derived from the region depicted in Frame 1, is illustrated so as to enhance the resolution. Glycosylated surfactant protein-A (SP-A) (gly), unglycosylated SP-A (ungly), and SP-A dimer (di) are labeled in the normal immunoblot (Frame 2). The immunochemical staining patterns were obtained using antibody raised against human surfactant Protein-A.

Under reducing conditions, glycosylated SP-A monomer and dimer constituted the major isoforms. Although the pattern of immunostaining was similar in lavage from RL (Figure 2) and LL, the proportion of high M_r nonreducible isoforms (Region A) varied between sides even though identical amounts of protein were loaded and that the paired samples were processed, separated, transferred, and stained in tandem. Moreover, additional isoforms were apparent in lavage fluid from some patients. Generally, these additional forms appeared in four distinct regions; B: 23 to 25 kD, 4.4 to 5.6 pI; C: 25 to 28 kD, 4.9 to 6.2 pI; D: ~ 45 kD, 5.1 to 5.5 pI; E: ~ 55 kD, 5.0 to 5.7 pI, although their relative abundance varied.

View larger version (62K): [in this window] [in a new window]   Figure 2.   Two-dimensional electrophoretic immunochemical staining patterns of whole lavages from matching right (RL) (Frames 1, 3, 5, and 7 ) and left (LL) (Frames 2, 4, 6, and 8) lungs of three typical patients with AP (Patient 1, Frames 1 and 2; Patient 2, Frames 3 and 4; Patient 3, Frames 5 and 6, lavage from the case study patient at the time of onset of the condition, and Frames 7 and 8, ~ 400 d later during the apparent resolution of the condition). Isoelectric focusing (IEF) of the samples was performed simultaneously. Only a selected window of immunochemical staining (Mr ~ 25 to ~ 100 kD and pI ~ 4.2 to ~ 6.4), derived from the region depicted in Figure 1 Frame 1, is illustrated and labeled in Frame 1 so as to enhance the resolution. Major regions of immunoreactivity have been labeled (glycosylated SP-A [gly], SP-A dimer [di], and A to D). The immunochemical staining patterns were obtained using antibody raised against human surfactant protein-A.

Case Study

On each occasion the 29-yr-old female patient was lavaged, the concentrations of DSP, CHOL, SP-A, SP-B, and PL (Figure 3) decreased proportionally with each sequential aliquot. Consequently, the %DSP/PL and the ratios CHOL/DSP, SP-A/ DSP, and SP-B/DSP remained the same in each aliquot.

View larger version (16K): [in this window] [in a new window]   Figure 3.   Changes in surfactant composition in each sequential aliquot during the whole lung lavage of the case study patient. The concentration is presented as the mean ± SEM of the six occasions that the right, and the five occasions that the left, lung was lavaged over ~ 13 mo. SP-A = surfactant protein-A; SP-B = surfactant protein-B; DSP = disaturated phospholipids; CHOL = free cholesterol.

When the serial aliquots for a given day were combined, whereas all ratios were greatly elevated on the first occasion the patient was lavaged, they progressively decreased towards normal values with a concomitant improvement in the patient's condition and the reduced need for lavage (Figure 4). These decreases were not due to changes in the absolute amount of DSP (Figure 4).

View larger version (15K): [in this window] [in a new window]   Figure 4.   Changes in the SP-A/DSP and SP-B/DSP ratios in lavage fluid derived from the right and left lung of the case study patient on separate occasions over ~ 13 mo. The ratios are presented as the mean ± SEM of the ~ 26 sequential aliquots collected on any lavage occasion. The DSP concentration (µg/ml) is that of the pooled lavage collected on that occasion. The normal SP-A/DSP (dotted line) and SP-B/DSP (dashed line) ratios are shown. Definition of abbreviations as in Figure 3.

SP-A Immunochemical Analysis

The patient's SP-A immunochemical staining patterns at the time of her entry into the study (Figure 2) were indistinguishable from those obtained ~ 400 d later. Left lung lavage continued to contain proportionally more of the high M_r nonreducible isoforms (Region A) than did RL lavage.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We have confirmed that the alveolar SP-A/DSP ratio is usually elevated in patients with AP and have shown for the first time that the same is equally true of SP-B/DSP. However, the relative abundance of SP-A and -B varies enormously between patients with AP. In addition, the relative abundance of the SP-A isoforms is consistently abnormal, but again it varies greatly between patients. In contrast, whereas the CHOL/DSP ratio was also greatly elevated, it was remarkedly constant between patients. There were only minor differences in the relative amounts of the surfactant phospholipids compared with that in normal subjects. Because of the inherent difficulties in standardizing the processing of lavage derived from various clinical settings, we did not attempt to determine whether the surfactant changes are specific to the tubular myelin-rich or myelin-poor alveolar fractions.

Surfactant Heterogeneity

Approximately 30% of AP cases resolve spontaneously, some require multiple lavage over extended periods, whereas others progress to disseminated lung disease (2). If left untreated ~ 30% of patients progress to dyspnea, hypoxemia, and death (2). This spectrum suggests multiple etiologies. Consistent with this, we found considerable variation in surfactant composition between patients, particularly with regard to the relative amounts of SP-A and -B. The high degree of internal consistency between RL and LL indicates that these differences are not artifacts. Although this begs the question as to the clinical course of the various patients, we only have complete data from the one case presented.

We found a decrease in the lavage PG/PI ratio, consistent with the suggestion of Honda and associates (1) that the condition is associated with a switching in the proportions of PI and PG synthesized from CDP-diacylglycerol. In addition, PI separated as a broad peak (not shown), consistent with changes in its fatty acid complement (1). Although similar changes have been observed in other respiratory diseases, including acute respiratory distress syndrome and idiopathic pulmonary fibrosis (1), their significance is unknown. The percentages of LPC and SPH were elevated in AP, consistent with increased surfactant degradation and cell damage, respectively (1).

Although the alveolar SP-A/DSP and SP-B/DSP ratios were markedly elevated in 12 of the patients, they were reduced in one. Possibly, this relates to disease severity, although we have no direct index of this. Importantly, the SP-B/SP-A ratio was normal in all patients. Recent studies suggest that granulocyte macrophage-colony stimulating factor (GM-CSF) may play a pivotal role in AP. GM-CSF has been localized in alveolar type II cells and in other respiratory epithelial cells, and it could be important in surfactant homeostasis (13). Mice deficient in GM-CSF develop respiratory disease morphologically and pathologically similar to AP (14), and a preliminary trial administering GM-CSF appears successful in treating an AP patient (15). Alveolar SP-A and -B are also elevated in mice deficient in GM-CSF, despite normal mRNA levels (14). The findings of Dranoff and coworkers (14) and Ikegami and colleagues (16) suggest that surfactant synthesis in GM-CSF mice is normal, at least with regard to saturated PC and SP-A and -B, and that the primary defect is impaired reuptake. This is consistent with the early studies in patients with AP by Ramirez-Rivera and Harlan (3) and McClenahan and Mussenden (4).

Both SP-A (5) and -B (17) stimulate the reuptake of surfactant lipids into type II cells, probably via separate high affinity receptors on the cell surface. Surfactant protein-B deficiency invariably results in a fatal congenital form of AP (18). Similarly, although mice deficient in SP-A do not exhibit any acute changes in surfactant turnover, DSP is increased, consistent with a small increase in phospholipid secretion and/or a small decrease in phospholipid uptake (19). Ikegami and associates (19) further estimate that a very small decrease in uptake at steady state would be sufficient to account for the 50% increase in the alveolar pool in 8-wk-old mice. Ueda and associates (20, 21) have reported that in rabbits the kinetics of SP-A (20) and -B (21) clearance are similar and approximately twice the rate of dipalmitoylphosphatidylcholine (DPPC). If the same is true in humans, and if indeed reuptake of surfactant lipids is mediated via SP-A and -B, then impaired uptake of both SP-A and -B would lead to an increase in the SP-A/PL and SP-B/PL ratios. Possibly, GM-CSF mediates the density of the SP-A (22) and -B receptors on the type II cell surface in a manner analogous to the action of secretagogues. However, an equally plausible explanation for the elevated alveolar SP-A and -B levels is that both proteins are functionally and structurally aberrant in AP (8, 9).

Surfactant Protein A

Our SP-A analysis confirms the findings of Voss and coworkers (8) and Hattori and colleagues (9) that AP lavage fluid contains a relatively large amount of the nonreducible protein. Although there were no obvious relationships between the abundance of the nonreducible forms and the patient's age, sex, or duration of AP, these may have become evident with additional patients. We report, for the first time, that the abundance of these high M_r forms clearly varied between RL and LL, possibly reflecting heterogeneity in the severity of the condition since their prevalence did not appear biased towards either a particular side or the side lavaged first. However, we do not have sufficient clinical data to clarify this further. We found additional lower M_r isoforms in four distinct regions (M_r: 23 to 25, 25 to 28, ~ 45, and ~ 55 kD). Again, their relative abundance varied markedly between patients.

SP-A is synthesized and is secreted by both the tracheobronchial epithelium, most likely from Clara cells, and the alveolar type II cells (23). Secreted SP-A displays extensive charge and mass heterogeneity, possibly reflecting alternative splicing of the mRNA transcript (6). Primary translation products of SP-A are further substantially and variably modified by the addition of complex asparagine-linked high-mannose oligosaccharides, which may in turn be trimmed by glucosidases and mannosidases or sialated, sulfated, and acetylated. In view of its complex quaternary structure and distinct binding domains, it is tempting to speculate that structural differences may contribute to the pathogenesis of AP. Hattori and coworkers (9) have shown that a subpopulation of AP SP-A fails to promote the formation of normal surfactant structures and suggest that this may disrupt the normal surfactant life cycle and promote accumulation in the alveolus. However, we believe that it is more likely that the aberrant SP-A forms arise through prolonged retention in the alveolus, possibly associated with reduced glutathione levels or exposure to oxidative enzymes or proteases, together with a possible impairment of the mucociliary escalator. The abundance of the aberrant SP-A forms vary markedly between patients. Both SP-A and -B are normally extensively post-translationally modified and secreted via distinct pathways that rely upon their structural integrity (5, 24). Given the elevated alveolar SP-A and -B levels, but normal SP-B/SP-A ratio, it is difficult to envisage a mechanism that gives rise to defects in both proteins without also affecting their secretion. Whatever the case, the variations we describe in SP-A derived from different patients with AP may, in part, explain some of the functional discrepancies reported (25).

Cholesterol

Cholesterol is normally the second most abundant lipid in surfactant after PC, comprising ~ 10% by weight and ~ 20 mol% (26). However, our analyses indicate that CHOL in AP lavage fluid is ~ 7-fold greater than normal, making it by far the most abundant component. Interestingly, whereas surfactant composition varied markedly with regard to the relative amounts of other constituents, the relative CHOL content was remarkedly constant between patients. Alveolar CHOL is purported to be derived primarily from serum lipoproteins via lamellar bodies (27). However, we have recently reported that lamellar bodies in fact contain little CHOL (12) and that CHOL and DPPC are handled independently (28). The role and source of CHOL in surfactant is unresolved. It may either enhance or reduce fluidity of phospholipids, depending on their phase transition temperature (29), and may therefore affect the rate of adsorption of newly released surfactant (26). Since it is likely that the protein and lipid components of surfactant are processed as aggregates (5), CHOL in AP may hinder reuptake by disrupting the phase transition temperature and aggregation of lipids destined for recycling.

Therapeutic Whole Lung Lavage

Although therapeutic lavage is standard treatment for relief of symptomatic AP, its effect on surfactant composition has never been studied. Progressive deterioration in oxygen saturation in patients with AP mandates therapeutic whole lung lavage. Surfactant recovery from one lavage procedure to the next will be determined, in part, not only by the severity of the condition but also by the time between the procedures. Whereas we have shown that sequential lavage does not selectively remove DSP, CHOL, SP-A, or SP-B, Alberti and associates (30) have recently reported differential changes in surfactant composition, including decreases in SP-A, 2 h after the procedure. They speculate that lavage removes factor(s) that otherwise interfere with the clearance of SP-A, and hence surfactant lipids, by type II cells. Possibly consistent with this, resolution of the condition in the case study patient appeared closely associated with the progressive normalization in the CHOL/DSP, SP-A/DSP, and SP-B/DSP ratios. Whether these changes were fortuitous or were the result of the multiple lavage performed during this period is unknown.

In conclusion, we have shown that although both SP-A and -B tend to be markedly elevated in AP, the SP-B/SP-A ratio is normal. The SP-A isoforms that make up the alveolar pool vary markedly between patients, and we suggest that caution must be used in comparing functional data derived using SP-A obtained from different patients with AP. Finally, the abundance of CHOL in AP lavage fluid clearly warrants further attention since it may directly impinge on surfactant reuptake.